PROJECT SUMMARY The increase in obesity worldwide has led to rising health care costs and a surge in diseases such as type 2 diabetes. This work will examine the molecular mechanism by which creatine metabolism regulates thermogenesis and protects from obesity and will test the idea that creatine metabolism protects against pancreatitis and ensuing diabetes. GATM is the rate-limiting enzyme of creatine synthesis. To examine the role adipose creatine in body weight regulation, I have generated mice with adipose-specific Gatm deletion (Adipo- Gatm KO). My preliminary data demonstrate that Adipo-Gatm KO mice have reduced energy expenditure, decreased body temperature upon exposure to environmental cold and profound susceptibility to diet-induced obesity. In the mentored phase of this award, Aim1 will identify unknown metabolic intermediates controlling creatine-dependent thermogenesis using a comparative metabolomics approach in vivo and stable 18O isotope tracer-based metabolomics in isolated mitochondria. Aim 2 of the mentored phase will delineate the protein factors mediating creatine-dependent thermogenesis using co-immunoprecipitation of mitochondrial Creatine Kinase (Mi-CK) and the ATP/ADP carrier (ANT) followed by quantitative proteomics. Aim 3 will be conducted in the independent R00 phase and will examine the role of creatine metabolism in protecting from pancreatitis and type 3c diabetes mellitus (T3cDM). This proposed research plan is a logical extension of my postdoctoral work and will be completed in the defined award period. I anticipate that my research findings will reveal novel metabolic intermediates and protein factors that can be targeted for obesity and diabetes therapeutics. My immediate career goal after the mentored phase of the K99 is to become an Assistant Professor at a leading academic research institute, where I will leverage my expertise in molecular metabolism to delineate novel pathways of thermogenic control by creatine. My long-term career goal is to establish myself as a leader in the field of molecular metabolism, energy balance, and obesity. Dr. Bruce Spiegelman, a well-recognized leader in the field of energy metabolism and obesity, will mentor my research career development. Numerous trainees of Dr. Spiegelman now hold prominent faculty positions. The Spiegelman laboratory and the Harvard Medical School research community provide an ideal setting for training towards independence. My plan for career activities includes continuous progress evaluation with my mentor, training in bioenergetics and mouse physiology, supervision and leadership training, educational coursework, and job search mentorship. These career activities will be essential as I prepare towards independence. My broad long-term research objective is to identify and target metabolic pathways that regulate energy balance in the context of obesity and diabetes. The NIH Pathway to Independence Award is fundamental for a successful transition because it will support my additional training in animal physiology, metabolomics and bioenergetics, and will facilitate my ability to improve my skills in scientific communication.